Cloning, Expression And Functional Analysis Of Antioxidant Genes In Roughskin Sculpin (Trachidermus Fasciatus)

Roughskin sculpin, Trachidermus fasciatus Heckel (Scorpaeniformes:Cottidae), is a carnivorous fish with seawater-freshwater migratory habits. As one of the second order of protected aquatic animals in China, roughskin sculpin is of great biological, ecological, and economic importance. Considering its serious decline during the last few decades, aquaculture has become an important strategy for recovering its population in the wild. However, roughskin sculpin possesses unique pathologic feature due to its unique biological and physiological characteristics. Up to now, few studies have reported on the molecular mechanisms of innate immunity in roughskin sculpin. It has been reported that reactive oxygen species (ROS) are highly produced in the process of microbial invasion or heavy metal exposure. ROS may be helpful to the host in the process of killing invading bacteria at low concentrations. On the other hand, the elevated levels of ROS could cause cytotoxicity to host cell and the immune system. Thus, to eliminate of these excessive ROS and maintain redox balance is of great importance for the proper function of cells and innate immune response of organisms.In the present study, thioredoxinl (designated TfTrxl), thioredoxin related protein14(designated TfTRP14) and peroxiredoxinl (designated TfPrxl) were cloned from roughskin sculpin using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time PCR (qRT-PCR) was employed to assess the mRNA expression of the three genes in various tissues and their temporal expression in important immune organs and brain of roughskin sculpin post E.coli LPS challenge and heavy metal exposure. To elucidate their biological functions, they were expressed in E. coli BL21(DE3).Trx is a small multifunctional protein ubiquitously expressed in bacteria, plant, and animals, which has a redox-active disulfide/dithiol within the conserved active site sequence CGPC (Cys-Pro-Asp-Cys). The full length cDNA of TfTrxl is665bp with a336bp ORF, encoding a protein of111amino acids with the predicted molecular weight of12.34kD and isoelectric point (pI) of4.52. No signal peptide was detected in the protein. The similarity was66%between roughskin sculpin and sablefish (Anoplopoma fimbria). The active sites CGPC were highly conserved according to the multiple sequence alignment. It has five β-sheets and four a-helixes, and the active sites locate on the surface of the protein. NJ tree reconstructed showed that TfTrx1clustered to Trxls from other fishes, in accordance to their traditional taxonomy. qRT-PCR analysis revealed the presence of TfTrx1transcripts in all detected tissues including blood, heart, liver, gill, stomach, intestine, skin, muscle, kidney, spleen, brain and ovary. The expression of TfTrx1mRNA in the skin, liver, spleen and brain was up-regulated post LPS challenge. In the skin and liver, the TfTrx1protein was significantly induced detected with western blot and maintained much longer time than the increase of TfTrx1mRNA. In the heavy metal exposure experiment, all the detected tissues (skin, liver, spleen, gill and brain) showed an increased expression. These results suggested that TfTrx1may be involved in the innate immunity related antioxidant response of roughskin sculpin. To investigate the biological activities of TfTrx1, a recombinant protein were constructed using an E. coli expression system. TfTrx1, which contains W34(Tryptophan, Trp or W), yielded a2.6-fold increase in tryptophan fluorencence when reduced, suggesting changes in the microenvironment around the W34residue. Furthermore, TfTrxl exhibited a concentration-dependent disulfide reductase activity towards insulin. These indicated that TfTrx1may play roles in fish immune response against oxidative damage in physiological state as a redox enzyme. In vitro incubation of TfTrx1with LPS demonstrated that TfTrx1could directly interact with E. coli LPS. Furthermore, TfTrx1could agglutination and binding gram-negative bacteria, possibly via the interaction with LPS. Thses suggested that TfTrx1may protect against LPS-induced tissue injury, and play role in the promoting phagacytosis of invaded pathogen.TRP14is a member of Trx superfamily with a Trx-like motif CPDC (Cys-Pro-Asp-Cys). The full length cDNA of TfTRP14is945bp with a372bp ORF. It was translated to a123amino acid protein with molecular weight of14.11kD and isoelectric point (pI) of5.45. The active sites CPDC were highly conserved from the multiple sequence alignment. The similarity was up to90%between roughskin sculpin and sablefish. It has five β-sheets and four α-helixes, the same as TfTrxl. However, the surface near the active sites of TfTRP14included an additional helix α3a and an extended β2-α2loop, which protruded, as characteristic of human TfTRP14. This may cause substrates difference between TRP14and Trx. TfTRP14was broadly expressed in all the examined tissues, with the highest transcript level in the brain and skin. The expression of TfTRP14was up-regulated post LPS challenge and heavy metal exposure, implying the involvement of TfTRP14in fish immune related antioxidant response. In addition, a fusion protein containing TfTRP14was produced in E. coli expression system. TfTRP14, which contained W42, W77yielded a5.4-fold increase in tryptophan fluorencence when reduced, higher than that of TfTrxl (2.6-fold), suggesting changes in the microenvironment around both W residues. In insulin reduction experiment, TfTRP14demonstrated a concentration-dependent disulfide reductase activity significantly lower than TfTrx1. This might be due to the substrates difference between these two proteins resulted from different redox characteristic.Prxs are abundant cellular antioxidative proteins which play important roles in protecting organisms against the toxicity of ROS. The full length cDNA of TfPrxl is1047bp with an ORF of600bp, encoding a protein of199amino acids. The predicted molecule weight is22.35kD and isoelectric point (pI) is6.42. Multiple sequence alignments demonstrated the presence of two characteristic peptides of Prx "FYPLDFTFVCPTEI" and "GEVCPA", indicated that TfPrxl was a classic2-Cys Prx. NJ tree showed that TfPrxl clustered with Prxls from other species, apart from Prx2s, indicating that rouskin sculpin Prx belonged to Prxl. TfPrx1ubiquitously expressed in all the detected tissues, with the most abundant expression in the intestine, followed by the brain. In the brain and main immune organs, the expression of TfPrxl was significantly induced post LPS chanllege and heavy metal exposure, suggesting that it play an important role in the innate immunity and antioxidant activity of roughskin sculpin. The recombinant protein of TfPrxl demonstrated the capability to reduce the H2O2when dithiothreitol (DTT) was used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that TfPrx1probably play an essential role in antioxidant defense in physiological state.